1. Field of the Invention
The present invention relates to neuronal culture media that may be used for high-throughput analyses.
2. Brief Description of the Related Art
The ability to maintain isolated neurons in primary culture has been critical in advancing our understanding of the functional basis of the nervous system. In primary neuronal culture studies, the use of defined culture conditions is essential for controlling the concentration of components, such as hormones and growth factors, which may affect the growth and development of cultured neurons.
Primary cultures of cerebellar neurons are an important in vitro model system that has been used because of their small size, the ease with which large numbers of cells can be isolated, and the resulting physical properties. Neonatal rat or mouse cerebellar granule cells are convenient and frequently used in studies with aims ranging from the identification of factors involved in neurogenesis, development, and migration to defining mechanisms of neurotoxicity and cell death (Cull-Candy, et al., J Physiol, 400: 189-222, 1988; Pemberton, et al. J Physiol, 510: 401-420, 1998; Bhave et al., J Neurosci, 19: 3277-3286, 1999). Because cultured granule cells express both Ca++ permeable-NMDA and non-NMDA glutamate receptors, these neurons are also an important model used for the analysis of neuronal excitotoxicity (Carroll et al., Neurochem Int, 33: 23-28, 1998; Lim and Ho, J Neurochem, 69: 581-593, 1998).
Over the past three decades, significant progress has been made in developing conditions for maintaining dissociated neurons in culture without the need for serum supplements. Media supplements, such as the relatively simple N2 medium supplement (Boftenstein and Sato, Proc Natl Acad Sci U.S.A. 76: 514-517, 1979) and the more complex neurobasal medium and B27 supplement (NB-B27) were originally designed for optimal viability of neurons in low-density primary culture systems (Brewer and Cotman, Brain Res, 494: 65-74, 1989; Brewer et al., J Neurosci Res, 35: 567-576, 1993; and Brewer, J Neurosci Res, 42: 674-683, 1995).
However, both the N2 and B27 supplements contain significant concentrations of the steroid hormone progesterone and B27 contains unspecified amounts of steroids, non-steroidal hormones, antioxidants, and other factors that may influence the outcome of neurotoxicity studies (Brewer, J Neurosci Res, 35: 567-576, 1995). It is well established that the activities of steroidal hormones can affect growth and proliferation mechanisms of cultured neurons (Singh et al., J Neurosci, 19: 1179-1188, 1999; Singh et al., J Neurosci, 19: 2455-2463, 1999; Toran-Allerand et al., Front Neuroendocrinol, 20: 97-121, 1999) as well as influence the functional properties of some neurotransmitter receptors (Smith, Brain Res, 503: 354-357, 1989; Smith, Brain Res Bull, 23: 317-322, 1989; Smith, Neurosci, 42: 309-320, 1991; Gu and Moss, J Neurosci, 16: 3620-3629, 1996; Gu et al., Endocrinology, 140: 660-666, 1999).
While Carroll et al., Neurochem Int, 33: 23-28 (1998), used NB-B27 in neurotoxicity studies of granule cells, the high concentrations of antioxidants contained in this complex media formulation may have masked the effects of many toxicants including excitotoxins and reactive oxygen species. U.S. Pat. Nos. 5,766,948 and 6,020,197 to Gage et al., which are incorporated herein by reference, disclose a medium that can be used both in neuroblast and cellular compositions. However, this medium is supplemented with trophic factors, a component specifically avoided in this invention.
Other studies such as those found in U.S. Pat. No. 6,040,180 to Johe, which is incorporated herein by reference, disclose using a serum-free medium supplemented with numerous growth factors. This culture media described in Johe, is used for generating differentiated neurons, but does not describe a media that can be used for toxicological studies.
Another component found in many types of growth media is phenol red. Phenol red, which is used primarily as a pH indicator, is very commonly used in tissue culture media, but can interfere with many assays. For example, U.S. Pat. No. 5,573,937 to Shinmoto et al., and U.S. Pat. No. 5,316,938 to Keen et al., which are incorporated herein by reference, both disclose media in which one of the components is phenol red.
Factors such as corticosterone, progesterone, retinyl acetate, T3 (triiodo-1-thyronine), xcex1-tocopherol (vitamin E) and phenol red which are present in NB-B27, may influence the outcome of studies assessing the effects of steroids on neuronal growth and development. U.S. Pat. No. 6,103,530 to Carpenter, which is incorporated herein by reference, discloses a culture medium for proliferating mammalian neural stem cells containing neural stem cell growth factors, such as transforming growth factor alpha, epidermal growth factor, or basic fibroblast growth factor. Likewise, U.S. Pat. No. 5,780,301 to Saito et al., also incorporated herein by reference, discloses a serum-free medium for culturing postnatal central neurons but includes the additional components of platelet-derived growth factors, vitronectin, and interleukin-1xcex2, all which may affect neuronal studies.
Studies used to measure neurotoxicity can be affected by added antioxidants in the growth media. Because antioxidants have been shown to efficiently reduce MTT (Liu and Schubert, J Neurochem, 69: 2285-2293, 1997) the high concentrations of antioxidants present in NB-B27 may compromise the usefulness of the MTT/MTS reduction assay for analysis of neurotoxicity in neuronal cultures maintained in NB-B27.
In addition to media that are serum-free, other investigators have used serum-containing media to support primary cultures of granule cells. The serum-containing media usually include undefined amounts of growth factors, steroids or other components that can influence viability, growth and differentiation of these cells. Ultimately, serum-containing media may not provide sufficiently well controlled study conditions for detection of modest, but biologically important, effects.
Each of these references describe a medium that contains extraneous components that may complicate studies aimed at elucidating the role of steroids during neuronal development, or how steroids regulate the functional properties of neurotransmitter receptors. As a result, studies aimed at defining factors that influence those processes are made more difficult when neurons are maintained in a culture medium containing undefined components. The limitations of the prior art are overcome by the present invention as described below. References mentioned in this background section are not admitted to be prior art with respect to the present invention.
The present invention is directed to satisfying the need for a medium capable of allowing growth and development of neuronal cultures, without containing components which may interfere with bioassays. The present invention describes, but is not limited to use in the MTS and lactate dehydrogenase bioassays.
An embodiment of the invention comprises maintaining cerebellar granule neurons in a simplified serum-free medium lacking exogenous antioxidants, growth factors, and steroids or steroid-like compounds in a versatile 96-well culture plate format. The present invention is not limited to a 96-well culture plate, and can be practiced with other tissue culture vessels.
Chemically defined culture conditions, free of added antioxidants, serum and steroid hormones are established for the growth, maintenance and analysis of primary cultured granule cells in 96-well tissue culture plates. Results indicate that granule cell viability can be maintained for at least 3 weeks in defined granule cell serum free (GCSF) medium that consists of HEPES buffered DMEM supplemented with insulin, transferrin, selenium and depolarizing concentrations of KCl (25 mM). Elevated K+ concentrations are used here because previous studies have indicated that the high concentrations of K+ or activation of NMDA receptors mediate transient increases in intracellular Ca++ concentrations that are required for maximal survival of cultured granule cells (Burgoyne et al., J Neurocytol., 22: 689-695, 1993).
The conditions set forth herein allows automated high-throughput analysis with the flexibility to employ many different bioassays, including the assessment of viability and cell death. Examples are given of the usefulness of these conditions with two methods frequently used to analyze neuronal toxicity; the MTT/MTS reduction assay to measure viability, and the LDH assay to measure cell death. The ability to easily maintain granule neurons under simple, well-defined conditions in 96-well plates provides a flexible and powerful model system that is suitable for rapidly determining the effects of various growth conditions, drugs, or toxicants on primary cultured neurons.